1. Field of the Invention
The present invention generally relates to a sample introduction method for guiding liquid samples into an analytical equipment, in particular, to a sample introduction method using a sample introduction device including a needle and an injection port connected to a flow path switching valve.
2. Description of Related Art
In order to analyze a plurality of samples, a sample introduction device for automatically guiding samples into an analytical equipment such as a liquid chromatograph in a specified sequence is adopted. FIG. 2(a) is a schematic view of a liquid chromatograph. The liquid chromatograph is formed of a liquid feeder 10, a sample introduction device 20, a separation/detection section 30, and a control/resolution section 40. The sample introduction device 20 is disposed between the liquid feeder 10 and the separation/detection section 30. The separation/detection section 30 includes an analysis column 31 and a detector 32, and various flow paths are formed for actual analysis purposes, thereby functioning as an analysis section. The liquid feeder 10, the sample introduction device 20, and the separation/detection section 30 are controlled by the control/resolution section 40. After receiving a signal from the detector 32, the control/resolution section 40 performs qualitative/quantitative resolution on the samples and saves the resolution data or files and outputs an analysis report.
The sample introduction device has the following two injection methods: a “total volume injection method” of injecting all measured samples from a sample container and a “partial injection method” of filling and injecting a part of the measured samples from the sample container into a sample loop (Patent Documents 1, 2, and Non-patent Document 1). In the fields that only quite a few samples can be collected, the total volume injection method is widely applied for analysis for not wasting the collected samples.
FIG. 2(b) is a schematic view of flow paths inside the sample introduction device 20 for the total volume injection method. The sample introduction device 20 forms the flow paths centered with a six-port two-position valve 21 and a six-position valve 22. A flow path of a mobile phase solution from the liquid feeder 10 into the sample introduction device 20 is first connected to one port of the six-port two-position valve 21. A flow path, which is from the upstream side of the liquid feeder 10 and a flow path, which is toward the downstream side of the separation/detection section 30 communicate with each other through a sample loop 23, a needle 24 disposed at a tip section of the sample loop 23 and an injection port 25 inserted with the needle 24. Therefore, all samples filled in the needle 24 to the sample loop 23 are guided into the separation/detection section 30. The six-position valve 22 is connected to a flow path in communication with a metering pump 26 for drawing a cleaning fluid from a cleaning fluid container or drawing samples from a sample container 28, and a flow path in communication with a cleaning port 27 provided for the insertion of the needle 24 so as to clean the needle. Moreover, the needle 24 and the sample loop 23 are communicated with the flow path of the metering pump 26 through the six-port two-position valve 21. In addition, the six-port two-position valve 21 switches the flow path of the mobile phase solution pressurized by the liquid feeder 10 and is thus called a “high pressure valve”. The six-position valve 22 is not connected to a flow path applied with a relatively high pressure and is thus called a “low pressure valve”. Accordingly, in this specification, the six-port two-position valve may be referred to as the “high pressure valve” and the six-position valve may be referred to as the “low pressure valve”.
To facilitate the understanding, a flow path switching valve, such as the high pressure valve 21 or the low pressure valve 22, is illustrated. In the flow path switching valve, a stator surface disposed with holes is joined to a rotor surface disposed with grooves, and each groove on the rotor surface (rotor groove) communicates with two holes on the stator surface (stator holes). The rotor rotates to make the rotor surface slide relative to the stator surface, so that a relative position between the rotor groove and the stator holes is changed, thereby switching a communication status between one stator hole and the other stator holes. Moreover, the stator holes are in communication with the ports disposed on the flow path switching valve respectively and each port is connected to a flow path. Therefore, when the rotor rotates to cause a change of the relative position between the rotor groove and the stator holes, a communication status of the flow path connected to the port is switched.
FIGS. 7(a) to 7(c) are diagrams respectively showing communication statuses of a joint surface of the high pressure valve 21 and the low pressure valve 22. The high pressure valve 21 in FIG. 7(a) is used for switching the flow paths in any of the two statuses. The high pressure valve 21 includes stator holes (a, b, c, d, e, and f) and arc-shaped rotor grooves (X, Y, and Z) centered with a rotation axis of the rotor. The high pressure valve 21 switches between a first status and a second status. In the first status, the rotor groove X communicates with the stator holes a and b, the rotor groove Y communicates with the stator holes c and d, and the rotor groove Z communicates with the stator holes e and f. In the second status, the rotor groove X communicates with the stator holes b and c, the rotor groove Y communicates with the stator holes d and e, and the rotor groove Z communicates with the stator holes f and a. In FIG. 2(b), in the high pressure valve 21, a port in communication with the stator hole a is connected to a flow path in communication with the needle 24 through the sample loop 23, a port in communication with the stator hole b is connected to a flow path in communication with the liquid feeder 10, a port in communication with the stator hole c is connected to a flow path in communication with the separation/detection section 30, and a port in communication with the stator hole d is connected to a flow path in communication with the injection port 25. The flow paths connected to a port in communication with the stator hole e and a port in communication with the stator hole f are determined according to the actual purposes and applications. When the high pressure valve 21 is in the first status, the flow path, which is from the upstream side of the liquid feeder 10 and the flow path, which is toward the downstream side of the separation/detection section 30 are communicated through the sample loop 23, the needle 24, and the injection port 25 (this status is also referred to as an “injection status”). When the high pressure valve 21 is in the second status, the flow path, which is from the upstream side of the liquid feeder 10 and the flow path, which is toward the downstream side of the separation/detection section 30 are not communicated through the sample loop 23, the needle 24, and the injection port 25 (this status is also referred to as a “load status”).
It takes tens of milliseconds to hundreds of milliseconds to switch between the first status and the second status. Generally, during this period, none of the stator holes are communicated, and in certain cases, a relatively long rotor groove is formed deliberately as mentioned in Non-patent Document 1. FIG. 7(b) depicts a high pressure valve in Non-patent Document 1, in which the rotor groove X is set longer than another rotor grooves Y and Z. The high pressure valve 21′ performs the following functions, which are using a metering pump to repeatedly draw and discharge samples and filling the samples in a sample loop having a volume greater than or equal to that of the metering pump while remaining in the load status. The high pressure valve 21′ is obtained through improvement on the structure disclosed in Patent Document 2.
The low pressure valve 22 in FIG. 7(c) is used for switching between six communication statuses, so as to enable a common port to be communicated with the other ports and/or enable various ports to be communicated with each other. The low pressure valve 22 includes stator holes (h, p, r, s, t, and u) and rotor grooves (V and W). The stator hole h is always connected to one end of the rotor groove V and is also in communication with the common port of the low pressure valve 22. The metering pump 26 or the cleaning port 27, the cleaning fluid container are connected to the other ports of the low pressure valve 22 and are also connected to the ports of the high pressure valve 21, so as to be in communication with the needle 24 and the injection port 25 etc. In FIG. 2(b), the common port in communication with the stator hole h is connected to the flow path connected with the metering pump 26. Samples are drawn or discharged from the sample container 28 and the cleaning fluid is drawn or discharged through the switching of the low pressure valve 22. Further, in order to accurately draw with the metering pump 26, the pressure in a sampling flow path (through the needle 24 and the sample loop 23) is at the atmospheric pressure, or other measures are taken. In addition, a low pressure valve without a common port is used in Non-patent Document 1.
Patent Document 1: Japanese Patent Publication No. H06-148157
Patent Document 2: Japanese Patent Publication No. H10-170488
Non-patent Document 1: “HPLC//LCtalk No. 46 TEC, INJECTION METHODS OF SAMPLE INTRODUCTION DEVICE (COMPARISON BETWEEN TOTAL VOLUME INJECTION METHOD AND PARTIAL INJECTION METHOD)”, Shimadzu Corporation, online, http://www.an.shimadzu.co.jp/support/lib/lctalk/46/46tec.htm, searched on Sep. 25, 2007.
In the total volume injection method shown in FIG. 2(b), when the high pressure valve 21 is in the load status, a specified volume of samples are drawn from the sample container 28 through the needle 24 and then filled into the sample loop 23 connected to a bottom section of the needle 24. Thereafter, the needle 24 is moved to the cleaning port 27 to have its outer surface cleaned. Afterward, the needle 24 is inserted into the injection port 25, and the high pressure valve 21 is switched to the injection status. The circulation of the mobile phase solution inside the sample loop 23 forces the samples filled in the sample loop 23 out and guides all the samples into the separation/detection section 30. For the guided samples, the high pressure valve remains in the injection status till the analysis is over. That is, during the analysis, the mobile phase solution keeps flowing inside the needle 24. In other words, the mobile phase solution remains in a cleaning status inside the needle 24.
Although the outer side of the needle 24 is cleaned at the cleaning port 27 and the inner side thereof is cleaned with the mobile phase solution, the problem of carry-over may still occur. The so-called carry-over means a phenomenon that a part of the injected samples are left behind and affect the next round of analysis. Although the carry-over is greatly alleviated through the surface treatment of the needle, the cleaning of the needle, and the change of the shape of the injection port, the problem still remains. Meanwhile, with the development of ultra-micro analysis and highly sensitive detection in recent years, the problem is growing worse. Therefore, the carry-over impedes the accurate analysis on the volume of samples drawn from the sample container 28.
After careful researches, the inventor of the present invention has identified the reason why carry-over still occurs even if the needle 24 is cleaned in the process of switching the high pressure valve from the load status to the injection status.
The switching of the flow paths is realized through the operation of the high pressure valve 21 and the processes for forming of the status in which the rotor grooves communicate with the stator holes are greatly related through the operation. As shown in FIG. 7(a), even if the three rotor grooves have the same length, the rotor surface and the stator surface sliding repeatedly may still be abraded due to long-time use; thus, the sections for forming the rotor grooves or the stator holes may be damaged. According to the different damaged sections, a status having the same effect as that formed with a relatively longer rotor groove is obtained. As a result, flow paths communicated in a time sequence different from the original one are generated. According to different damaged sections and the degrees of the damage, the high pressure valve 21 becomes a valve the same as shown in FIG. 7(b). As described above, the high pressure valve in FIG. 7(b) is applicable for processing samples having a large volume (from hundreds of μl to several ml) exceeding a 1-stroke volume of the metering pump 26, but is not suitable for processing a minute volume of the samples.
FIGS. 6(a) to 6(d) show the flowing directions of the samples at a circumference of an insertion section of the needle 24 and the injection port 25 as well as the movement of the samples inside the needle 24 in a period from the moment that the high pressure valve 21′ is switched to the injection status immediately after the needle 24 is inserted into the injection port 25 till the moment that all the samples flow to the downstream side. FIGS. 6(a) to 6(d) also show a generation mechanism of carry-over caused by the abraded high pressure valve 21′.
First of all, FIG. 6(a) shows a status that the needle 24 is inserted into the injection port 25 after the sample solution is measured. Till the high pressure valve 21′ is switched from the load status to the injection status, the samples are located at a tip section inside the needle 24 and the needle 24 is filled with the mobile phase solution in a manner of holding the samples.
Referring to FIG. 6(b), during the switching from the load status to the injection status, only the rotor groove X communicates with the stator holes a and b among the three rotor grooves, enabling the liquid feeder 10 and the needle 24 to communicate with each other. At this time, a part of the samples are forced out of the tip section of the needle 24 into the injection port 25 under the pressure of the liquid feeder 10. In this case, as the injection port 25 does not communicate with the separation/detection section 30, and the samples cannot flow to the downstream side, so that the part of the samples under the pressure of the liquid feeder are forced into a gap between the tip section of the needle 24 and the injection port 25 (FIG. 6(c)). The section marked by a circle in FIG. 6(c) is an amplified view of the tip section of the needle. A gap exists between the tip section of the needle 24 after taper machining and an inner wall of the injection port 25 substantially formed perpendicularly thereto, so that the samples are forced into the gap.
Afterward, the injection port 25 is in communication with the separation/detection section 30, and the samples are guided into the separation/detection section 30 under the influence of the mobile phase solution delivered by the liquid feeder 10. However, the portions of the samples that are forced into the gap are not guided into the separation/detection section 30, but are left in the injection port 25 instead (FIG. 6(d)). That is, as for the carry-over that still occurs even if the needle 24 is cleaned, during the switching from the load status to the injection status, the communication between the needle 24 and the liquid feeder 10 (the stator holes a and b communicate with each other through the rotor groove X) results in carry-over more easily, when compared with the communication between the injection port 25 and the separation/detection section 30 (the stator holes c and d communicate with each other through the rotor groove Y).